Hippo and dystrophin complex signaling in cardiomyocyte renewal

ABSTRACT

Embodiments of the disclosure include methods and compositions for the renewal of cardiomyocytes by targeting the Hippo pathway. In particular embodiments, an individual with a need for cardiomyocyte renewal is provided an effective amount of a shRNA molecule that targets the Sav1 gene. Particular shRNA sequences are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. NonProvisional patentapplication Ser. No. 16/544,209 filed on Aug. 19, 2019, which is acontinuation of U.S. NonProvisional patent application Ser. No.16/164,550 filed on Oct. 18, 2018, which is a continuation of U.S.NonProvisional patent application Ser. No. 15/642,200 filed on Jul. 5,2017 and registered as U.S. Pat. No. 10,119,141 on Nov. 6, 2018, whichis a continuation of U.S. NonProvisional patent application Ser. No.15/102,593 filed on Jun. 8, 2016 and registered as U.S. Pat. No.9,732,345 on Aug. 15, 2017, which is a national phase application under35 U.S.C. § 371 of International Application No. PCT/US2014/069349,filed Dec. 9, 2014, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/913,715, filed Dec. 9, 2013, the entire contentsof each are incorporated by reference herein their entirety.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under Grant HL007676awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present disclosure concerns at least the fields of cell biology,molecular biology, and medicine.

BACKGROUND

Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD), a lethal inherited X-linked disorderoccurring in 1 of every 3500 male births (Emery, 2002), is characterizedby rapid and progressive degeneration of skeletal and cardiac musclefibers. Importantly, DMD patients develop heart disease marked bymyocardial necrosis, fibrosis and dilated cardiomyopathy. DMD arisesfrom mutation of the dystrophin gene that encodes a 427 kd cytoskeletalprotein present in skeletal, cardiac and smooth muscle cells (Hoffman etal., 1987; Hoffman et al., 1988). In DMD patients, dystrophin expressionis abolished, leading to disruption of the dystrophin-associatedglycoprotein complex (DGC), an essential membrane localized structure inskeletal and cardiac muscle (Ohlendieck and Campbell, 1991; Ohlendiecket al., 1993). A valuable mouse model for DMD is the mdx mouse, adystrophin-null strain that exhibits a disease phenotype with similarityto human DMD (Bulfield et al., 1984). Although much less severe thanhuman DMD, the mdx mice have characteristics of the human disease suchas skeletal muscle degeneration/regeneration and cardiomyopathy afteraging.

Introduction to Hippo-Signaling

The mammalian core Hippo-signaling components include the Ste20 kinasesMst1 and Mst2 that are orthologous to the Drosophila Hippo kinase. Mstkinases, when complexed with the Salvador (Salv) scaffold protein,phosphorylate the Large Tumor Suppressor Homolog (Lats) kinases.Mammalian Lats1 and Lats2 are NDR family kinases and are orthologous toDrosophila Warts. Lats kinases, in turn, phosphorylate Yap and Taz, tworelated transcriptional co-activators that are the most downstreamHippo-signaling components and partner with transcription factors suchas Tead to regulate gene expression. Yap also interacts with β-catenin,an effector of canonical Wnt signaling to regulate gene expression. Uponphosphorylation, Yap and Taz are excluded from the nucleus and renderedtranscriptionally inactive (FIG. 1).

Previous cardiac loss-of-function studies in mice revealed thatHippo-signaling inhibits cardiomyocyte proliferation to control heartsize (Heallen et al., 2011). Salv deficient hearts develop cardiomegalywith a 2.5-fold increase in heart size due to cardiomyocyte hyperplasia.Additionally, experiments investigating Yap in cardiomyocyte developmentsupport the conclusion that Yap is the major Hippo effector moleculeduring cardiomyocyte development (von Gise et al., 2012; Xin et al.,2011). Yap is a cofactor that partners with DNA binding transcriptionalregulators. The current literature indicates that Tead-family co factorsare primary Yap partners (Halder and Johnson, 2011).

The present disclosure concerns methods and compositions that address along-felt need in the art to provide therapy for cardiac conditions,including at least DMD, by targeting the Hippo pathway.

BRIEF SUMMARY

The present invention is directed to methods and compositions thatprovide therapy for at least one medical condition that directly orindirectly affects cardiac muscle cells (cardiomyocytes) in a mammalianindividual, including humans, dogs, cats, horses, pigs, and so forth.The medical condition may be of any kind, including a cardiac conditionsuch as heart failure, cardiomyopathy, myocardial infarction, and soforth. The medical condition may have a cardiac condition as its primarysymptom or cause or it may be a secondary symptom or cause. Theindividual may be male or female and may be of any age.

In particular embodiments, an individual in need of therapy for acardiac medical condition is provided an effective amount of one or morenucleic acids, or cells comprising one or more nucleic acids, in whichthe nucleic acids provide therapeutic benefit to the individual. Inspecific embodiments, the nucleic acid is a form that directly orindirectly provides RNA interference, including at least shRNA. Inparticular embodiments, the shRNA composition targets a member of theHippo pathway. Although it may target any member of the Hippo pathway,in specific embodiments, the shRNA targets Salvador (Sav1).

In embodiments of the disclosure, there are nucleic acid compositionsthat target Sav1 of a mammal, and in specific embodiments the nucleicacid compositions are shRNA molecules. In specific embodiments, thereare therapeutic compositions that comprise shRNA molecules comprisingone or more of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Thecompositions may or may not be encompassed in a vector, including aviral vector or non-viral vector. In particular embodiments, the shRNAsequences are utilized in a non-integrating vector.

SUMMARY

In some embodiments, there is an isolated synthetic nucleic acidcomposition, comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ IDNO:12 and/or a derivative nucleic acid comprising at least 80% identityto one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Thederivative nucleic acid may be at least 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ IDNO:12.

In certain embodiments, the nucleic acid comprises the sequence of SEQID NO:4 (or SEQ ID NO:5, 6, 7, 8, 9, 10, 11, or 12) and furthercomprises an antisense sequence of SEQ ID NO:4 (or, respectively, SEQ IDNO:5, 6, 7, 8, 9, 10, 11, or 12), wherein when the sequence and theantisense sequence are hybridized together to form a duplex structure,the sequence and the antisense sequence are separated by a loopstructure.

In specific embodiments, the nucleic acid is at least 43 nucleotides inlength or no more than 137 nucleotides in length. In some embodiments,the loop structure is between 5 and 19 nucleotides in length. Inparticular embodiments, the derivative nucleic acid has 1, 2, 3, 4, or 5mismatches compared to the respective SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,or SEQ ID NO:12.

In some embodiments of the composition, the nucleic acid or derivativenucleic acid is comprised in a vector, such as a viral vector or anon-viral vector. The vector may be a non-integrating vector. The vectormay be a non-integrating vector that is a lentiviral vector. In someembodiments of the vector, two or more of nucleic acids comprising SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 are present on thesame vector.

In specific embodiments, the expression of the nucleic acid is regulatedby a tissue-specific or cell-specific promoter, such as acardiomyocyte-specific promoter, for example the rat ventricle-specificcardiac myosin light chain 2 (MLC-2v) promoter; cardiac muscle-specificalpha myosin heavy chain (MHC) gene promoter; cardiac cell-specificminimum promoter from −137 to +85 of NCX1 promoter; chicken cardiactroponin T (cTNT), or a combination thereof.

In certain embodiments, two or more of nucleic acids comprising SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 are present on the samevector. In specific cases, two or more nucleic acids are regulated bythe same regulatory sequence or are regulated by a different regulatorysequence.

In one embodiment, there is a method of treating an individual for acardiac condition, comprising the step of providing an effective amountof a composition encompassed by the disclosure to the individual. Incertain embodiments, the cardiac condition in the individual causes theindividual to be in need of cardiomyocyte renewal. In certainembodiments, the heart of the individual has cardiomyocyte apoptosis,necrosis, and/or autophagy. In specific embodiments, the cardiaccondition comprises cardiovascular disease, cardiomyopathy, heartfailure, myocardial infarction, ischemia, necrosis, fibrosis, ordiabetic cardiomyopathy, age-related cardiomyopathy. In particularembodiments, the individual has Duchenne muscular dystrophy. Thecomposition may be provided to the individual more than once. Thecomposition may be provided to the individual systemically or locally.In a specific embodiment, the individual is provided an additionaltherapy for the cardiac condition.

In certain embodiments, there is a kit comprising a composition asencompassed by the disclosure, wherein the composition is housed in asuitable container.

In particular embodiments, there is a method of treating a cardiaccondition in an individual, comprising the step of providing to theindividual a therapeutically effective amount of a shRNA that targetsSalvador (Sav1). In a specific embodiment, the shRNA is provided to theindividual in the AAV9 vector. In particular cases, the individual hasDuchenne muscular dystrophy.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 illustrates an example of a model for Hippo signaling duringdevelopment and regeneration;

FIGS. 2A and 2B shows that Yap regulates cell division and motilitygenes in cardiac regeneration. Heat maps (FIG. 2A) and qRT-PCRvalidation (FIG. 2B) showing relative transcript levels for a subset ofYap targets. *p<0.05,**p<0.001, ***p<0.0001. Adapted from (Morikawa etal., 2014);

FIG. 3 shows Salv CKO and Salv CKO; mdx; utrn hearts after apexresection. Hearts were resected at postnatal day 8, a non-regenerativestage, and evaluated by immunohistochemistry at three weeks afterresection. The Salv CKO mutant hearts can regenerate as reported (lefthand panel and (Heallen et al., 2013). In addition, the compound mutantSalvCKO; mdx−; utrn+/−hearts can also regenerate as shown in the panelon the right. Notably, mdx mutant hearts fail to regenerate aftercardiomyocyte resection;

FIG. 4 demonstrates that Hippo depletion promotes cardiac functionalrecovery after chronic ischemia and established heart failure. Adulthearts were subjected to LAD-O (t=0) and studied by echocardiographythree weeks later. Hearts that were in failure were entered into thestudy and tamoxifen was injected to inactivate Salv. Mice were studiedby echocardiography at two-week intervals as shown. Hippo deficienthearts showed recovery of function at two-weeks post tamoxifen injectionand by six weeks functional recovery was complete;

FIG. 5 shows that Salv siRNA effective reduces Salv mRNA expression.siRNAs were transfected into neonatal cardiomyocytes and Salv mRNAlevels were studied by quantitative RT PCR. All three siRNAs effectivelyreduced Salv mRNA levels;

FIG. 6 illustrates the mouse Sav1 cDNA sequence (SEQ ID NO:1). Thegrayed regions demonstrate examples of shRNA sequences (SEQ ID NO:4, SEQID NO:5, and SEQ ID NO:6, respectively). Alternating exons are show bysequences that are double-underlined vs. sequences that are notdouble-underlined. Protein structural domains are shown by sequencesthat are single underlined, in order of 5′ to 3′; WW domain, WW domain,SARAH domain.

FIG. 7 illustrates the pig Sav1 cDNA sequence (SEQ ID NO:2). The grayedregions demonstrate examples of shRNA sequences (SEQ ID NO:7, SEQ IDNO:8, and SEQ ID NO:9, respectively). Alternating exons are show bysequences that are double-underlined vs. sequences that are notdouble-underlined. Protein structural domains are shown by sequencesthat are single underlined, in order of 5′ to 3′; WW domain, WW domain,SARAH domain; and

FIG. 8 illustrates the human Sav1 cDNA sequence (SEQ ID NO:3). Thegrayed regions demonstrate examples of shRNA sequences (SEQ ID NO:10,SEQ ID NO:11, and SEQ ID NO:12, respectively). Alternating exons areshow by sequences that are double-underlined vs. sequences that are notdouble-underlined. Protein structural domains are shown by sequencesthat are single underlined, in order of 5′ to 3′; WW domain, WW domain,SARAH domain.

DETAILED DESCRIPTION I. Exemplary Definitions

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

As used herein, the term “complementary nucleotide sequence,” also knownas an “antisense sequence,” refers to a sequence of a nucleic acid thatis completely complementary to the sequence of a “sense” nucleic acidencoding a protein (e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an mRNA sequence).Herein, nucleic acid molecules are provided that comprise a sequencecomplementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 nucleotides.

As used herein, the term “correspond to a nucleotide sequence” refers toa nucleotide sequence of a nucleic acid encoding an identical sequence.In some instances, when antisense nucleotides (nucleic acids) or siRNA's(small inhibitory RNA) (processed from the shRNA) bind to a targetsequence a particular antisense or small inhibitory RNA (siRNA) sequenceis substantially complementary to the target sequence, and thus willspecifically bind to a portion of an mRNA encoding polypeptide. As such,typically the sequences of those nucleic acids will be highlycomplementary to the mRNA target sequence, and will have no more than 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence.In many instances, it may be desirable for the sequences of the nucleicacids to be exact matches, i.e. be completely complementary to thesequence to which the oligonucleotide specifically binds, and thereforehave zero mismatches along the complementary stretch. Highlycomplementary sequences will typically bind quite specifically to thetarget sequence region of the mRNA and will therefore be highlyefficient in reducing, and/or even inhibiting the translation of thetarget mRNA sequence into polypeptide product. See, for example, U.S.Pat. No. 7,416,849.

Substantially complementary oligonucleotide sequences will be greaterthan about 80 percent complementary (or ‘% exact-match’) to thecorresponding mRNA target sequence to which the oligonucleotidespecifically binds, and will, more preferably be greater than about 85percent complementary to the corresponding mRNA target sequence to whichthe oligonucleotide specifically binds. In certain aspects, as describedabove, it will be desirable to have even more substantiallycomplementary oligonucleotide sequences for use in the practice of theinvention, and in such instances, the oligonucleotide sequences will begreater than about 90 percent complementary to the corresponding mRNAtarget sequence to which the oligonucleotide specifically binds, and mayin certain embodiments be greater than about 95 percent complementary tothe corresponding mRNA target sequence to which the oligonucleotidespecifically binds, and even up to and including 96%, 97%, 98%, 99%, andeven 100% exact match complementary to the target mRNA to which thedesigned oligonucleotide specifically binds. See, for example, U.S. Pat.No. 7,416,849. Percent similarity or percent complementary of anynucleic acid sequence may be determined, for example, by utilizing anycomputer programs known in the art.

As used herein, the term “knock-down” or “knock-down technology” refersto a technique of gene silencing in which the expression of a targetgene or gene of interest is reduced as compared to the gene expressionprior to the introduction of the shRNA, which can lead to the inhibitionof production of the target gene product. The term “reduced” is usedherein to indicate that the target gene expression is lowered by0.1-100%. For example, the expression may be reduced by 0.5, 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, oreven 99%. The expression may be reduced by any amount (%) within thoseintervals, such as for example, 2-4, 11-14, 16-19, 21-24, 26-29, 31-34,36-39, 41-44, 46-49, 51-54, 56-59, 61-64, 66-69, 71-74, 76-79, 81-84,86-89, 91-94, 96, 97, 98 or 99. Knock-down of gene expression can bedirected by the use of siRNAs or shRNAs.

As used herein, the term “nucleotide sequence” refers to a polymer ofDNA or RNA which can be single-stranded or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The term “polynucleotide” isused interchangeably with the term “oligonucleotide.” The term“nucleotide sequence” is interchangeable with “nucleic acid sequence”unless otherwise clearly stated. “Nucleotide sequence” and “nucleic acidsequence” are terms referring to a sequence of nucleotides in apolynucleotide molecule.

As used herein, the term “operably-linked” refers to the association ofnucleic acid sequences on a polynucleotide so that the function of oneof the sequences is affected by another. For example, a regulatory DNAsequence is said to be “operably linked to” a DNA sequence that codesfor an RNA (“an RNA coding sequence” or “shRNA encoding sequence”) or apolypeptide if the two sequences are situated such that the regulatoryDNA sequence affects expression of the coding DNA sequence (i.e., thatthe coding sequence or functional RNA is under the transcriptionalcontrol of the promoter). Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation. An RNA codingsequence refers to a nucleic acid that can serve as a template forsynthesis of an RNA molecule such as an siRNA and an shRNA. Preferably,the RNA coding region is a DNA sequence.

As used herein, the term “pharmaceutically acceptable” is a carrier,diluent, excipient, and/or salt that is compatible with the otheringredients of the formulation, and not deleterious to the recipientthereof. The active ingredient for administration may be present as apowder or as granules; as a solution, a suspension or an emulsion or asdescribed elsewhere throughout the specification.

As used herein, the term “promoter” refers to a nucleotide sequence,usually upstream (5′) to its coding sequence, which directs and/orcontrols the expression of the coding sequence by providing therecognition for RNA polymerase and other factors required for propertranscription. “Promoter” includes a minimal promoter that is a shortDNA sequence comprised of a TATA-box and other sequences that serve tospecify the site of transcription initiation, to which regulatoryelements are added for control of expression. “Promoter” also refers toa nucleotide sequence that includes a minimal promoter plus regulatoryelements that is capable of controlling the expression of a codingsequence or functional RNA. This type of promoter sequence consists ofproximal and more distal upstream elements, the latter elements oftenreferred to as enhancers. Accordingly, an “enhancer” is a DNA sequencethat stimulates promoter activity and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. It is capable of operating in bothorientations (sense or antisense), and is capable of functioning evenwhen moved either upstream or downstream from the promoter. Bothenhancers and other upstream promoter elements bind sequence-specificDNA-binding proteins that mediate their effects. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven be comprised of synthetic DNA segments. A promoter may also containDNA sequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions. Any promoter known in the artwhich regulates the expression of the shRNA or RNA coding sequence isenvisioned in the practice of the invention.

As used herein, the term “reporter element” or “marker” is meant apolynucleotide that encodes a polypeptide capable of being detected in ascreening assay. Examples of polypeptides encoded by reporter elementsinclude, but are not limited to, lacZ, GFP, luciferase, andchloramphenicol acetyltransferase. See, for example, U.S. Pat. No.7,416,849. Many reporter elements and marker genes are known in the artand envisioned for use in the inventions disclosed herein.

As used herein, the term “RNA transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.“Messenger RNA transcript (mRNA)” refers to the RNA that is withoutintrons and that can be translated into protein by the cell.

As used herein, the terms “small interfering” or “short interfering RNA”or “siRNA” refer to an RNA duplex of nucleotides that is targeted to adesired gene and is capable of inhibiting the expression of a gene withwhich it shares homology. The RNA duplex comprises two complementarysingle-stranded RNAs of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides that form 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 basepairs and possess 3′ overhangs of two nucleotides. The RNA duplex isformed by the complementary pairing between two regions of a RNAmolecule. siRNA is “targeted” to a gene in that the nucleotide sequenceof the duplex portion of the siRNA is complementary to a nucleotidesequence of the targeted gene. In some embodiments, the length of theduplex of siRNAs is less than 30 nucleotides. The duplex can be 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10nucleotides in length. The length of the duplex can be 17-25 nucleotidesin length. The duplex RNA can be expressed in a cell from a singleconstruct.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex wherein a portion of the siRNA is part of a hairpin structure(shRNA). In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments the loop is5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpinstructure can also contain 3′ or 5′ overhang portions. In some aspects,the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides inlength. In one aspect of this invention, a nucleotide sequence in thevector serves as a template for the expression of a small hairpin RNA,comprising a sense region, a loop region and an antisense region.Following expression the sense and antisense regions form a duplex. Itis this duplex, forming the shRNA, which hybridizes to, for example, theSav1 mRNA and reduces expression of Sav1.

As used herein, the term “treating” refers to ameliorating at least onesymptom of, curing and/or preventing the development of a disease ordisorder such as for example, but not limited to, ischemic heartdisease, heart failure, cardiomyopathy, etc.

As used herein, the term “vector” refers to any viral or non-viralvector, as well as any plasmid, cosmid, phage or binary vector in doubleor single stranded linear or circular form that may or may not beself-transmissible or mobilizable, and that can transform prokaryotic oreukaryotic host cells either by integration into the cellular genome orwhich can exist extrachromosomally (e.g., autonomous replicating plasmidwith an origin of replication). Any vector known in the art isenvisioned for use in the practice of this invention.

II. General Embodiments

Embodiments of the disclosure concern methods and compositions fortreatment of cardiac medical conditions, including those in whichcardiomyocytes are in need of being renewed. The cardiomyocytes may bein need of renewal for any reason, including for disease, underlyinggenetic condition, and/or trauma, for example. In specific embodiments,the individual has cardiomyocyte injury, necrosis, and/or fibrosis ofthe heart, such as with Duchenne muscular dystrophy (DMD), for example.

In specific embodiments, methods and compositions are employed for anindividual with DMD. The diseased heart in DMD patients has widespreadcardiomyocyte injury, necrosis and fibrosis. Similarly, mdx mutant micethat model human DMD have cardiomyopathy with heart failure and severefibrosis and dilation. The Hippo signaling pathway was identified as acritical repressor of cardiac regeneration following tissue amputationor myocardial infarction (Heallen et al., 2013). In addition, dataindicate that Hippo regulates transcription of DMD-related genes in theheart. Taken together, it was considered that Hippo signaling inhibits arepair response to Duchenne cardiomyopathy. As shown herein, neonatalmdx hearts fail to regenerate following apex resection, in contrast toregenerative wild-type neonate hearts. Most notably, this regenerativecapacity was largely restored in Hippo; mdx compound mutant hearts,indicating genetic suppression of the mdx heart phenotype. Takentogether, modulating Hippo signaling serves as a powerful approach torepair heart muscle, such as DMD heart muscle.

In addition to improving DMD cardiomyopathy, it is shown herein thatHippo pathway inhibition after established heart failure (HF)drastically improved cardiac function in a mouse model of ischemiccardiomyopathy and HF. Mice with established ischemic cardiomyopathy andHF were generated by waiting three weeks after induction of a myocardialinfarct to deplete the Hippo pathway. At the three-week time point, theHippo pathway was inactivated and cardiac function was followed attwo-week intervals by echocardiography. Hippo deficiency stronglyenhances cardiac repair in the context of established HF such that Hippomutants recover function equal to that of un-operated sham controls.

There is demonstrated herein a unique, but exemplary, set of three shorthairpin RNAs (shRNA) that specifically target the Hippo pathway memberSalvador (Sav1). The shRNAs provide selective reduction in Sav1 mRNAlevels similar to a genetic knockout in a mouse model. In specificembodiments, the shRNAs can be delivered using an AAV9 (Adeno AssociatedVirus serotype 9) vector that has tropism for the heart. Particularembodiments of the disclosure contemplate the shRNA sequence ofnucleotides specific to target Sav1.

III. Salvador

In particular embodiments, the Hippo pathway member Salvador (salvadorfamily WW domain containing protein 1) is targeted with shRNA intreatments for cardiac medical conditions. The gene may be referred toas salvador homolog 1, Salv, SAV1, SAV, WW45, or WWP4. A representativenucleic acid is provided at GenBank® Accession No. CR457297.1, and arepresentative protein sequence is provided at GenBank® Accession No.Q9H4B6.

The gene encodes a protein which includes 2 WW domains (a modularprotein domain that mediates specific interactions with protein ligands)and a coiled-coil region. It is ubiquitously expressed in adult tissues.It also includes a SARAH (Sav/Rassf/Hpo) domain at the C terminus (threeclasses of eukaryotic tumor suppressors that give the domain its name)In the Say (Salvador) and Hpo (Hippo) families, the SARAH domainmediates signal transduction from Hpo via the Say scaffolding protein tothe downstream component Wts (Warts); the phosphorylation of Wts by Hpotriggers cell cycle arrest and apoptosis by down-regulating cyclin E,Diap 1 and other targets. The SARAH domain may also be involved indimerization.

IV. Examples of/Methods of Treatment

In embodiments of the disclosure, there are methods of treating anindividual with a cardiac condition using nucleic acids that target theSav1 gene. In specific embodiments, the cardiac condition includescardiomyocytes that are in need of renewal either because of disease(contracted or genetic, for example) or because of trauma, for example.In specific embodiments, there is diseased heart in the individual. Theindividual may have cardiomyocytes that are in need of renewal for anyreason. Cardiomyocytes in the individual may be apoptotic, autophagic,or the tissue may be necrotic, for example.

In specific embodiments, the individual may have heart failure, fibrosisof the heart, cardiomyopathy, ischemic cardiomyopathy, myocardialnecrosis, dilated cardiomyopathy, degeneration of skeletal and/orcardiac muscle fibers, Diabetic cardiomyopathy, age-relatedcardiomyopathy, and so forth. In specific embodiments, methods of thedisclosure allow for the ability of cardiomyocytes to re-enter the cellcycle. The individual may be in need of improved cardiac function forany reason, including because of age, disease, trauma, and so forth.

In particular embodiments, the individual is provided an effectiveamount of nucleic acid that targets Sav1 such that existingcardiomyocytes in the individual are able to renew. In otherembodiments, an individual is provided nucleic acids that target Sav1wherein the nucleic acids are already present in a cell at the time ofdelivery, including a cardiomyocyte or stem cell, for example.

The nucleic acid compositions of the disclosure may be provided to theindividual once or more than once. The delivery may occur upon thediagnosis of a need for cardiomyocyte renewal or upon diagnosis of acardiac condition. Delivery may occur to an individual who issusceptible to a cardiac condition, such as having a personal or familyhistory, being overweight, having high cholesterol, and/or a smoker. Thedelivery may cease or continue once it is determined that a cardiacsymptom is improved and/or that cardiomyocytes are being renewed.

V. Nucleic Acids that Target Sav1

In particular embodiments, there are one or more nucleic acids thattarget Sav1 such that expression of Sav1 is detectably reduced. Thenucleic acids may be DNA or RNA, but in specific embodiments the nucleicacids are RNA, such as shRNA.

In one embodiment, the shRNA is a “hairpin” or stem-loop RNA molecule,comprising a sense region, a loop region and an antisense regioncomplementary to the sense region. In other embodiments the shRNAcomprises two distinct RNA molecules that are non-covalently associatedto form a duplex. See, for example, U.S. Pat. No. 7,195,916.

In particular cases, shRNA is a single-stranded RNA molecule that formsa stem-loop structure in vivo, and it may be from about 40 to 135nucleotides in length. In at least certain cases, a 5- to 19-nucleotideloop connects the two complementary 19- to 29-nucleotide-long RNAfragments that create the double-stranded stem by base pairing.Transcription and synthesis of shRNA in vivo is directed by Pol IIIpromoter, and then the resulting shRNA is cleaved by Dicer, an RNase IIIenzyme, to generate mature siRNA. The mature siRNA enters the RISCcomplex. Thus, in specific embodiments, shRNA for inhibition of Sav1expression in accordance with the present disclosure contains both senseand antisense nucleotide sequences.

Although the present disclosure provides specific examples ofSav1-targeting shRNAs (SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, or 12), othershRNA compositions may be employed. Those of skill in the art mayidentify appropriate sequences in any manner, but in specificembodiments one can align the gene from two or more organisms, scanoverlapping regions for the amino acid-encoding sequence, review thesequence for regions of a certain length (such as, for example 19 nt),review the sequence for those having no more than 3 nt repeats, and/orblast potential sequences to ensure there is <15 bp homology to anyother part of the human genome.

When appropriately targeted via its nucleotide sequence to a specificmRNA in cells, the shRNA specifically suppress gene expression of Sav1.In at least some cases, shRNAs can reduce the cellular level of specificmRNAs, and decrease the level of proteins coded by such mRNAs. shRNAsutilize sequence complementarity to target an mRNA for destruction, andare sequence-specific. Thus, they can be highly target-specific, and inmammals have been shown to target mRNAs encoded by different alleles ofthe same gene.

In specific embodiments, an shRNA corresponding to a region of a targetgene to be down-regulated or knocked-down is expressed in the cell. TheshRNA duplex may be substantially identical (for example, at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) in sequence to the sequenceof the gene targeted for down regulation. In specific embodiments, thereare no more than 5 mismatches between the sequence of the shRNA and thetarget Sav1 sequence. In specific embodiments, a minimum of 18 bphomology is utilized for the region of complementarity between the shRNAsequence and its target. In particular embodiments, specific assays areutilized to test suitable mismatches for the shRNA and its target. Incertain embodiments, an algorithm may be employed to identify suitablemismatches for the shRNA and its target.

Thus, it should be noted that full complementarity between the targetsequence and the shRNA is not required. That is, the resultant antisensesiRNA (following processing of the shRNA) is sufficiently complementarywith the target sequence. The sense strand is substantiallycomplementary with the antisense strand to anneal (hybridize) to theantisense strand under biological conditions.

In particular, the complementary polynucleotide sequence of shRNA can bedesigned to specifically hybridize to a particular region of a desiredtarget protein or mRNA to interfere with replication, transcription, ortranslation. The term “hybridize” or variations thereof, refers to asufficient degree of complementarity or pairing between an antisensenucleotide sequence and a target DNA or mRNA such that stable andspecific binding occurs there between. In particular, 100%complementarity or pairing is desirable but not required. Specifichybridization occurs when sufficient hybridization occurs between theantisense nucleotide sequence and its intended target nucleic acids inthe substantial absence of non-specific binding of the antisensenucleotide sequence to non-target sequences under predeterminedconditions, e.g., for purposes of in vivo treatment, preferably underphysiological conditions. Preferably, specific hybridization results inthe interference with normal expression of the gene product encoded bythe target DNA or mRNA.

For example, an antisense nucleotide sequence can be designed tospecifically hybridize to the replication or transcription regulatoryregions of a target gene, or the translation regulatory regions such astranslation initiation region and exon/intron junctions, or the codingregions of a target mRNA. In specific embodiments, the shRNA targets asequence that encodes the N-terminal region of the Sav1 protein,sequence that encodes the middle of the Sav1 protein, or sequence thatencodes the C-terminal region of the Sav1 protein.

shRNA: Synthesis

As is generally known in the art, commonly used oligonucleotides areoligomers or polymers of ribonucleic acid or deoxyribonucleic acidhaving a combination of naturally-occurring purine and pyrimidine bases,sugars and covalent linkages between nucleosides including a phosphategroup in a phosphodiester linkage. However, it is noted that the term“oligonucleotides” also encompasses various non-naturally occurringmimetics and derivatives, i.e., modified forms, of naturally-occurringoligonucleotides as described below.

shRNA molecules of the disclosure can be prepared by any method known inthe art for the synthesis of DNA and RNA molecules. These includetechniques for chemically synthesizing oligodeoxy-ribonucleotides andoligo-ribonucleotides well known in the art such as, for example, solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the shRNA molecule. Such DNA sequences may be incorporated intoa wide variety of vectors that incorporate suitable RNA polymerasepromoters such as the T7 or SP6 polymerase promoters. Alternatively,antisense cDNA constructs that synthesize shRNA constitutively orinducibly, depending on the promoter used, can be introduced stably intocell lines.

shRNA molecules can be chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. Custom shRNA synthesis services are available fromcommercial vendors such as Ambion (Austin, Tex., USA) and DharmaconResearch (Lafayette, Colo., USA). See, for example, U.S. Pat. No.7,410,944.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.An antisense nucleic acid of the invention can be constructed usingchemical synthesis or enzymatic ligation reactions using proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally-occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids (e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used).

The shRNA molecules of the invention can be various modified equivalentsof the structures of any Sav1 shRNA. A “modified equivalent” means amodified form of a particular siRNA molecule having the sametarget-specificity (i.e., recognizing the same mRNA molecules thatcomplement the unmodified particular siRNA molecule). Thus, a modifiedequivalent of an unmodified siRNA molecule can have modifiedribonucleotides, that is, ribonucleotides that contain a modification inthe chemical structure of an unmodified nucleotide base, sugar and/orphosphate (or phosphodiester linkage). See, for example, U.S. Pat. No.7,410,944.

Preferably, modified shRNA molecules contain modified backbones ornon-natural internucleoside linkages, e.g., modifiedphosphorous-containing backbones and non-phosphorous backbones such asmorpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate,sulfonamide, and sulfamate backbones; formacetyl and thioformacetylbackbones; alkene-containing backbones; methyleneimino andmethylenehydrazino backbones; amide backbones, and the like. See, forexample, U.S. Pat. No. 7,410,944.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See, for example, U.S.Pat. No. 7,410,944.

Examples of the non-phosphorous containing backbones described above areknown in the art, e.g., U.S. Pat. No. 5,677,439, each of which is hereinincorporated by reference. See, for example, U.S. Pat. No. 7,410,944.

Modified forms of shRNA compounds can also contain modified nucleosides(nucleoside analogs), i.e., modified purine or pyrimidine bases, e.g.,5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), 2-thiouridine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyl uridine,5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine,3-methylcytidine, propyne, quesosine, wybutosine, wybutoxosine,beta-D-galactosylqueosine, N-2, N-6 and 0-substituted purines, inosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, N6-methyladenosine,7-methylguanosine, 2-methylthio-N-6-isopentenyl adenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives, and the like. See, for example, U.S. Pat. No.7,410,944.

In addition, modified shRNA compounds can also have substituted ormodified sugar moieties, e.g., 2′-O-methoxyethyl sugar moieties. See,for example, U.S. Pat. No. 7,410,944.

Additionally, to assist in the design of shRNAs for the efficientsilencing of any target gene, several supply companies maintainweb-based design tools that utilize these general guidelines for“picking” shRNAs when presented with the mRNA or coding DNA sequence ofthe target gene. Examples of such tools can be found at the web sites ofDharmacon, Inc. (Lafayette, Colo.), Ambion, Inc. (Austin, Tex.). As anexample, selecting shRNAs involves choosing a site/sequence unique tothe target gene (i.e., sequences that share no significant homology withgenes other than the one being targeted), so that other genes are notinadvertently targeted by the same shRNA designed for this particulartarget sequence.

Another criterion to be considered is whether or not the target sequenceincludes a known polymorphic site. If so, shRNAs designed to target oneparticular allele may not effectively target another allele, sincesingle base mismatches between the target sequence and its complementarystrand in a given shRNA can greatly reduce the effectiveness of RNAiinduced by that shRNA. Given that target sequence and such design toolsand design criteria, an ordinarily skilled artisan apprised of thepresent disclosure should be able to design and synthesized additionalsiRNA compounds useful in reducing the mRNA level of Sav1.

shRNA: Administration

The present disclosure provides a composition of a polymer or excipientand one or more vectors encoding one or more shRNA molecules. The vectorcan be formulated into a pharmaceutical composition with suitablecarriers and administered into a mammal using any suitable route ofadministration.

Because of this precision, side effects typically associated withtraditional drugs can be reduced or eliminated. In addition, shRNA arerelatively stable and, like antisense, they can also be modified toachieve improved pharmaceutical characteristics, such as increasedstability, deliverability, and ease of manufacture. Moreover, becauseshRNA molecules take advantage of a natural cellular pathway, i.e., RNAinterference, they are highly efficient in destroying targeted mRNAmolecules. As a result, it is relatively easy to achieve atherapeutically effective concentration of an shRNA compound in asubject. See, for example, U.S. Pat. No. 7,410,944.

shRNA compounds may be administered to mammals by various methodsthrough different routes. They can also be delivered directly to aparticular organ or tissue by any suitable localized administrationmethods such as direct injection into a target tissue. Alternatively,they may be delivered encapsulated in liposomes, by iontophoresis, or byincorporation into other vehicles such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres.

In vivo inhibition of specific gene expression by RNAi injectedintravenously has been achieved in various organisms including mammals.In particular embodiments, the shRNA molecules are comprised in avector, including a viral or non-viral vector. In specific embodiments,the vector is non-integrating, although in other embodiments it isintegrating. Viral vectors may be lentiviral, adenoviral,adeno-associated viral, and retroviral, for example. Non-viral vectorsinclude plasmids. In specific embodiments, the AAV9 vector (Piras etal., 2013) is employed. Vectors may be delivered to an individualsystemically or locally. In certain embodiments, the vectors utilizetissue-specific or cell-specific promoters, such ascardiomyocyte-specific promoters. In specific embodiments, the vectorsare delivered by local injection.

One route of administration of shRNA molecules of the invention includesdirect injection of the vector at a desired tissue site, such as forexample, into diseased cardiac tissue or into ischemic heart tissue.

In general, included in the invention is a vector comprising apolynucleotide sequence, and a promoter operably-linked to an isolatednucleic acid sequence encoding a first segment, a second segment locatedimmediately 3′ of the first segment, and a third segment locatedimmediately 3′ of the second segment, wherein the first and thirdsegments are each less than 30 base pairs in length and each more than10 base pairs in length, and wherein the sequence of the third segmentis the complement of the sequence of the first segment. The secondsegment, located immediately 3′ of the first segment, encodes a loopstructure containing from 4-10 nucleotides (i.e., 4, 5, 6, 7, 8, 9, 10).The nucleic acid sequence is expressed as an siRNA and functions as asmall hairpin RNA molecule (shRNA) targeted against a designated nucleicacid sequence.

More specifically, the present invention includes compositions andmethods for selectively reducing the expression of the gene product fromSav1. The present invention provides a vector comprising apolynucleotide sequence which comprises a nucleic acid sequence encodinga shRNA targeted against Sav1. The shRNA forms a hairpin structurecomprising a duplex structure and a loop structure. The loop structuremay contain from 4 to 10 nucleotides, such as 4, 5 or 6 nucleotides. Theduplex is less than 30 nucleotides in length, such as from 10 to 27nucleotides. The shRNA may further comprise an overhang region. Such anoverhang may be a 3′ overhang region or a 5′ overhang region. Theoverhang region may be, for example, 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The invention provides, inter alia, a method of treating a mammal byadministering to the mammal a composition comprising one or more vectorsdescribed herein. In one aspect of the invention, multiple vectors eachencoding a different shRNA (targeted to a different region of the Sav1nucleic acid sequence) may be administered simultaneously orconsecutively to the mammal. An individual vector may encode multipleshRNAs targeted to different areas of the same gene; i.e., comprisingtwo or more of a shRNA comprising SEQ ID NO: 10 and shRNA comprising SEQID NO: 11 and a shRNA comprising SEQ ID NO: 12. In another aspect, anindividual vector may encode multiple copies of shRNA comprising SEQ IDNO: 10 or multiple copies of shRNA comprising SEQ ID NO: 11 or multiplecopies of shRNA comprising SEQ ID NO: 12, in any ratio.

The vector of the invention may further comprise a promoter. Examples ofpromoters include regulatable promoters and constitutive promoters. Forexample, the promoter may be a CMV or RSV promoter. The vector mayfurther comprise a polyadenylation signal, such as a synthetic minimalpolyadenylation signal. Many such promoters are known in the art and areenvisioned for use in this invention. In other instances, the promotermay be a tissue specific promoter, such as a cardiac tissue specificpromoter.

The vector may further comprise one or more marker genes or reportergenes. Many marker genes and reporter genes are known in the art. Thepresent invention contemplates use of one or more marker genes and/orreporter genes known in the art in the practice of the invention. Themarker genes or reporter genes provide a method to track expression ofone or more linked genes. The marker genes or reporter genes uponexpression within the cell, provide products, usually proteins,detectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. Gene expression products, whetherfrom the gene of interest, marker genes or reporter genes may also bedetected by labeling. Labels envisioned for use in the inventionsincluded herein include, but are not limited to, fluorescent dyes,electron-dense reagents, enzymes (for example, as commonly used in anELISA), biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide. See, forexample, U.S. Pat. No. 7,419,779.

In one aspect of the invention, one or more vectors comprising one ormore of shRNA of the invention can be re-administered an unlimitednumber of times after a first administration at any time interval orintervals after the first administration.

shRNA: Pharmaceutical Compositions

The shRNA encoding nucleic acids of the present invention can beformulated in pharmaceutical compositions, which are prepared accordingto conventional pharmaceutical compounding techniques. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). The pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of the vector encodingshRNA. These compositions can comprise, in addition to the vector, apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The carrier can take a wide variety of forms depending onthe form of preparation desired for administration, e.g., intravenous,oral, intramuscular, subcutaneous, intrathecal, epineural or parenteral.

When the vectors of the invention are prepared for administration, theymay be combined with a pharmaceutically acceptable carrier, diluent orexcipient to form a pharmaceutical formulation, or unit dosage form. Thetotal active ingredients in such formulations include from 0.1 to 99.9%by weight of the formulation

In another aspect of the invention, the vectors of the invention can besuitably formulated and introduced into the environment of the cell byany means that allows for a sufficient portion of the sample to enterthe cell to induce gene silencing, if it is to occur. Many formulationsfor vectors are known in the art and can be used so long as the vectorsgain entry to the target cells so that it can act.

For example, the vectors can be formulated in buffer solutions such asphosphate buffered saline solutions comprising liposomes, micellarstructures, and capsids. The pharmaceutical formulations of the vectorsof the invention can also take the form of an aqueous or anhydroussolution or dispersion, or alternatively the form of an emulsion orsuspension. The pharmaceutical formulations of the vectors of thepresent invention may include, as optional ingredients, solubilizing oremulsifying agents, and salts of the type that are well-known in theart. Specific non-limiting examples of the carriers and/or diluents thatare useful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable saline solutions. Otherpharmaceutically acceptable carriers for preparing a composition foradministration to an individual include, for example, solvents orvehicles such as glycols, glycerol, or injectable organic esters. Apharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the shRNA encoding vector. Other physiologicallyacceptable carriers include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative or synthetic origin.The carrier can also contain other ingredients, for example,preservatives.

It will be recognized that the choice of a pharmaceutically acceptablecarrier, including a physiologically acceptable compound, depends, forexample, on the route of administration of the composition. Thecomposition containing the vectors can also contain a second reagentsuch as a diagnostic reagent, nutritional substance, toxin, oradditional therapeutic agent. Many agents useful in the treatment ofcardiac disease are known in the art and are envisioned for use inconjunction with the vectors of this invention.

Formulations of vectors with cationic lipids can be used to facilitatetransfection of the vectors into cells. For example, cationic lipids,such as lipofectin, cationic glycerol derivatives, and polycationicmolecules, such as polylysine, can be used. Suitable lipids include, forexample, Oligofectamine and Lipofectamine (Life Technologies) which canbe used according to the manufacturer's instructions.

Suitable amounts of vector must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual vector species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, or compositionsin which concentrations of about 1 nanomolar or less can be used. Inother aspects, the methods utilize a concentration of about 200picomolar or less and even a concentration of about 50 picomolar or lesscan be used in many circumstances. One of skill in the art can determinethe effective concentration for any particular mammalian subject usingstandard methods.

The shRNA is preferably administered in a therapeutically effectiveamount. The actual amount administered, and the rate and time-course ofadministration, will depend on the nature and severity of the condition,disease or disorder being treated. Prescription of treatment, forexample, decisions on dosage, timing, etc., is within the responsibilityof general practitioners or specialists, and typically takes account ofthe disorder, condition or disease to be treated, the condition of theindividual mammalian subject, the site of delivery, the method ofadministration and other factors known to practitioners. Examples oftechniques and protocols can be found in Remington's PharmaceuticalSciences 18th Ed. (1990, Mack Publishing Co., Easton, Pa.).

Alternatively, targeting therapies can be used to deliver the shRNAencoding vectors more specifically to certain types of cell, by the useof targeting systems such as antibodies or cell specific ligands.Targeting can be desirable for a variety of reasons, e.g., if the agentis unacceptably toxic, or if it would otherwise require too high adosage, or if it would not otherwise be able to enter the target cells.

shRNA: Gene Therapy

siRNA can also be delivered into mammalian cells, particularly humancells, by a gene therapy approach, using a DNA vector from which siRNAcompounds in, e.g., small hairpin form (shRNA), can be transcribeddirectly. Recent studies have demonstrated that while double-strandedsiRNAs are very effective at mediating RNAi, short, single-stranded,hairpin-shaped RNAs can also mediate RNAi, presumably because they foldinto intramolecular duplexes that are processed into double-strandedsiRNAs by cellular enzymes. This discovery has significant andfar-reaching implications, since the production of such shRNAs can bereadily achieved in vivo by transfecting cells or tissues with DNAvectors bearing short inverted repeats separated by a small number of(e.g., 3, 4, 5, 6, 7, 8, 9) nucleotides that direct the transcription ofsuch small hairpin RNAs. Additionally, if mechanisms are included todirect the integration of the vector or a vector segment into thehost-cell genome, or to ensure the stability of the transcriptionvector, the RNAi caused by the encoded shRNAs, can be made stable andheritable. Not only have such techniques been used to “knock down” theexpression of specific genes in mammalian cells, but they have now beensuccessfully employed to knock down the expression of exogenouslyexpressed transgenes, as well as endogenous genes in the brain and liverof living mice.

Gene therapy is carried out according to generally accepted methods asare known in the art. See, for example, U.S. Pat. Nos. 5,837,492 and5,800,998 and references cited therein. Vectors in the context of genetherapy are meant to include those polynucleotide sequences containingsequences sufficient to express a polynucleotide encoded therein. If thepolynucleotide encodes an shRNA, expression will produce the antisensepolynucleotide sequence. Thus, in this context, expression does notrequire that a protein product be synthesized. In addition to the shRNAencoded in the vector, the vector also contains a promoter functional ineukaryotic cells. The shRNA sequence is under control of this promoter.Suitable eukaryotic promoters include those described elsewhere hereinand as are known in the art. The expression vector may also includesequences, such as selectable markers, reporter genes and otherregulatory sequences conventionally used.

Accordingly, the amount of shRNA generated in situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the nucleic acid sequence encoding a shRNA sequence that are in thecell.

For expression of Sav1 shRNA, a promoter is operatively linked to ashRNA sequence. As used herein, the term “promoter” refers to a DNAsequence that regulates expression of the target gene sequence beingoperatively linked to the promoter sequence in a certain host cell. Theterm “operatively linked” means that one nucleic acid fragment is linkedto another nucleic acid fragment so that the function or expressionthereof is affected by the other nucleic acid fragment. The expressioncassette of the present invention may further comprise variousexpression regulatory sequences such as an optional operator sequencefor controlling transcription, a sequence encoding a suitable mRNAribosome-binding site, and sequences controlling the termination oftranscription and translation. The promoter used in the presentinvention may be a constitutive promoter that constitutively induces theexpression of a target gene, or an inducible promoter that induces theexpression of a target gene at a given position and time point. Specificexamples of the promoter may include U6 promoter, CMV (cytomegalovirus)promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene,108:193-199, 1991; and Monahan et al., Gene Therapy, 7:24-30, 2000),CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), Rsyn7promoter (U.S. patent application Ser. No. 08/991,601), ubiquitinpromoter (Christensen et al., Plant Mol. Biol. 12:619-632, 1989), ALSpromoter (U.S. patent application Ser. No. 08/409,297) and the like.Also usable promoters are disclosed in U.S. Pat. Nos. 5,608,149,5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463,5,608,142, etc.

The recombinant vector of the present disclosure may be introduced intoa host cell, using a conventional method known in the art. The host cellmay be employed for manipulation of the vector or as a means to transferthe vector to an individual. Preferably, intracellular incorporation ofthe vector into the host cell may be carried out by a conventionalmethod known in the art, such as calcium chloride, microprojectilebombardment, electroporation, PEG-mediated fusion, microinjection,liposome-mediated method, and the like.

Examples of the host cell that can be utilized in the present inventionmay include, but are not limited to, prokaryotic cells such asEscherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteusmirabilis, and Staphylococcus, lower eukaryotic cells such as fungi(e.g. Aspergillus), yeast (e.g. Pichia pastoris), Saccharomycescerevisiae, Schizosaccharomyces, and Neurospora crassa, and highereukaryotic cells such as insect cells, plant cells, mammalian cells.Preferably, the host cell may be human cells.

Meanwhile, standard recombinant DNA and molecular cloning techniquesused in the present disclosure are well known in the art and can befound in the following literature: Sambrook, J., Fritsch, E. F. andManiatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); Silhavy, T.J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); andAusubel, F. M. et al., Current Protocols in Molecular Biology, publishedby Greene Publishing Assoc. and Wiley-Interscience (1987).

The pharmaceutical composition according to the present invention maycomprise a therapeutically effective amount of the recombinant vector ofthe present invention and a cardiac drug alone or in combination withone or more pharmaceutically acceptable carriers. As used herein, theterm “therapeutically effective amount” refers to an amount which iscapable of producing the desired therapeutic response greater than thatexhibited by a negative control. Preferably, the therapeuticallyeffective amount is a dose sufficient to prevent or treat thecardiovascular disease.

A therapeutically effective amount of the recombinant vector in thepresent disclosure may be in a range of 0.0001 to 100 mg/day/kg (BW),preferably 0.005-0.05 mg/day/kg. However, an effective dose of the drugmay vary depending upon various factors such as kinds and severity ofdisease, age, weight, health and sex of patients, administration routesand treatment duration.

As used herein, the term “pharmaceutically acceptable” means that thecompound is physiologically acceptable, and does not cause allergicreactions (such as gastrointestinal disorders, and vertigo) or similarreactions with no inhibitory effects on the action of an activeingredient, when it is administered to humans or animals. Examples ofthe pharmaceutically acceptable carrier may include all kinds ofsolvents, dispersion media, oil-in-water or water-in-oil emulsions,aqueous compositions, liposomes, microbeads and microsomes.

Meanwhile, the pharmaceutical composition of the present invention maybe appropriately formulated in conjunction with any suitable carrier bya conventional method known in the art, depending upon administrationroutes of the drug. There is no particular limit to the administrationroute of the pharmaceutical composition. Therefore, the drug compositionin accordance with the present invention may be administered via oral orparenteral routes. Examples of the parenteral administration route mayinclude transdermal, intranasal, intraperitoneal, intramuscular,subcutaneous and intravenous routes.

When the pharmaceutical composition of the present invention isadministered via an oral route, the pharmaceutical composition inconjunction with any orally acceptable vehicle may be formulated intovarious dosage forms such as powders, granules, tablets, pills, dragees,capsules, solutions, gels, syrups, suspensions, and wafers, according toa conventional method known in the art. Examples of suitable vehiclesmay include various kinds of fillers, for example sugars such aslactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol andmaltitol; starches such as corn starch, wheat starch, rice starch andpotato starch; cellulose substances such as cellulose, methyl cellulose,sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose;gelatin, polyvinylpyrrolidone (PVP) and the like. If desired, there maybe added disintegrating agents such as cross-linkedpolyvinylpyrrolidone, agar, and alginic acid or sodium alginate.Further, the pharmaceutical composition may further compriseanticoagulants, lubricants, wetting agents, fragrances, emulsifiers andpreservatives.

When the pharmaceutical composition of the present invention isadministered via a parenteral route, the pharmaceutical composition inconjunction with any parenterally acceptable vehicle may be formulatedinto, for example, an injectable preparation, a transdermal preparationor a nasal inhalant, according to a conventional method known in theart. Upon formulation of the injectable preparation, sterilization mustbe performed in conjunction with protection of the pharmaceuticalpreparation from microbial contamination including pathogenic bacteriaand fungi. Examples of the vehicle suitable for the injectablepreparation may include, but are not limited to, solvents or dispersionmedia including water, ethanol, polyols (such as glycerol, propyleneglycol, and liquid polyethylene glycol), mixtures thereof and/orvegetable oil. More preferably, examples of the suitable vehicle mayinclude isotonic solutions such as Hank's solution, Ringer's solution,PBS (phosphate buffered saline) containing triethanolamine, sterilewater for injection, 10% ethanol, 40% propylene glycol and 5% dextrose.In order to protect the injectable preparation against microbialcontamination, the preparation may further comprise variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugar or sodium chloride.

In the case of the transdermal formulation, the inventive pharmaceuticalcomposition may be formulated in the form of ointments, creams, lotions,gels, external solutions, pastes, liniments, or aerosols. The term“transdermal administration” means that a therapeutically effectiveamount of an active ingredient contained in a pharmaceutical compositiontransmits into the skin when the pharmaceutical composition is topicallyapplied to the skin. These formulations are described in the literaturethat is a guidebook generally known in all pharmaceutical chemistryfields (Remington's Pharmaceutical Sciences, 15.sup.th Edition, 1975,Mack Publishing Company, Easton, Pa.).

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gases. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powdered mixture of the compound and a suitable powder basesuch as lactose or starch.

Other pharmaceutically acceptable vehicles can be found in theliterature (Remington's Pharmaceutical Sciences, 19.sup.th ed., MackPublishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention may furthercomprise one or more buffers (e.g. saline or PBS), carbohydrates (e.g.glucose, mannose, sucrose or dextran), antioxidants, bacteriostaticagents, chelating agents (e.g. EDTA or glutathione), adjuvants (e.g.aluminum hydroxide), suspending agents, thickening agents, and/orpreservatives.

Additionally, the pharmaceutical composition of the present inventionmay be appropriately formulated by a conventional method known in theart, such that it is possible to achieve fast, sustained or delayedrelease of active ingredients after administration of the composition toa mammal.

Further, the pharmaceutical composition of the present invention may beadministered in combination with a known drug having therapeutic effectsfor treating a cardiac condition.

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention

Example 1 Hippo Signaling Promotes Adult Cardiomyocyte Renewal

While other organs have regenerative capacity, cardiomyocytes fail torenew or to regenerate sufficiently to repair the damaged heart (Kikuchiand Poss, 2012). To investigate the hypothesis that Hippo-signaling is anegative regulator of postnatal cardiomyocyte renewal, we inactivatedSalv and both Lats genes in adult cardiomyocytes.

To test the role of Salv, Lats1, and Lats2 in adult cardiomyocytes,conditional null alleles were used for Hippo genes and theMyh6^(creERT2) transgene that directs tamoxifen-regulated cardiomyocytecre activity (Sohal et al., 2001). Because the heart contains multiplecell types, cardiomyocytes were visualized using the R26^(mTmG) (mTmG)allele, which expresses eGFP upon cre activation, to trace thecardiomyocyte lineage (Muzumdar et al., 2007). Adult cardiomyocytes weregenerated that were mutant for Salv and Lats1/2 by injecting threemonth-old mice with tamoxifen (Heallen et al., 2013). To determine ifHippo deficiency results in cell cycle reentry, mice were injected with5-ethynyl-2′-deoxyuridine (EdU). Nuclear EdU incorporation, indicatingde novo DNA synthesis, was detected in both Salv conditional knock out(CKO) and Lats1/2 CKO mutant cardiomyocytes revealing an endogenouscardiomyocyte renewal capacity when Hippo-signaling is deleted.Quantification of EdU positive cells showed significant induction of DNAsynthesis in Hippo-deficient hearts with a greater increase in Lats1/2mutants compared to Salv CKO cardiomyocytes (Heallen et al., 2013). Cellcycle reentry was also quantified in isolated cardiomyocyte nuclei usingFACS analysis (Bergmann et al., 2009; Heallen et al., 2013). BothLats1/2 CKO and Salv CKO cardiomyocyte nuclei had increased numbers ofKi-67 expressing cardiomyocytes compared to controls (Heallen et al.,2013). These results show that cardiomyocytes re-enter the cell cycleupon Hippo pathway disruption, supporting the hypothesis thatHippo-signaling is a negative regulator of adult cardiomyocyte renewal.

It was evaluated whether Salv CKO and Lats1/2 CKO cardiomyocytesprogress through mitosis and cytokinesis Immunohistochemistry wasperformed with the M-phase marker Aurora B kinase (Aurkb) to determineif cytokinesis occurred in Hippo-deficient cardiomyocytes. Aurkbexpression in Lats1/2 and Salv CKO cardiomyocytes was clearly detectableat the cleavage furrow providing direct evidence for cytokinesis(Heallen et al., 2013). In contrast to Hippo-deficient hearts, Aurkbexpression was not detected in control hearts.

In summary, Hippo pathway inactivation in the unstressed adult mouseheart results in enhanced cardiomyocyte renewal with increasedmyocardial S-phase entry and progression through mitosis (Heallen etal., 2013). These findings uncover an inhibitory role forHippo-signaling in adult cardiomyocyte renewal.

Example 2 Hippo Signaling Promotes Adult Heart Regeneration in an AcuteInjury Model

Cardiac apex resection in the first six days of life results in cardiacregeneration while resections performed at postnatal day (P) 7 and laterresults in fibrosis and scarring (Porrello et al., 2011).

To test regenerative capability, apex resection was performed of uniformsize at the normally non-regenerative P8 stage in control andHippo-deficient hearts. To inactivate Salv, mice were injected with fourtamoxifen doses prior to and after the resection. Both GFP fluorescence,detecting recombination in the mTmG reporter, and immunofluorescencewith an anti-Salv antibody indicated efficient deletion of Salv inmutant myocardium at four days post resection (4 dpr). Evaluation of 21dpr hearts by serial sectioning revealed severe scarring of controlhearts in all but a few cases. In contrast, resected Hippo-deficienthearts efficiently regenerated the myocardium with reduced scar size(Heallen et al., 2013). The regenerated cardiac apex was derivedprimarily from pre-existing cardiomyocytes.

Left anterior descending (LAD) coronary artery occlusion was performedat both P8 and two months of age. In P8 hearts, following LAD occlusion(LADO) there was functional recovery and reduced scar size when analyzedat twenty one days after occlusion. Histology also confirmed therecovery of myocardium with less scar tissue after LADO (Heallen et al.,2013). In adult hearts, there was similarly strong histologic andfunctional evidence for cardiomyocyte regeneration after LADO.Fractional Shortening and Ejection Fraction evaluated byechocardiography indicated that by three weeks post LADO, adultHippo-deficient hearts had recovered function comparable to that of shamoperated animals suggesting that Hippo-deficient cardiomyocytes haveincreased survival and/or proliferation after ischemic damage (Heallenet al., 2013).

Example 3 Hippo-Deficient Cardiomyocytes Extensively Proliferate andAcquire Migratory Properties During Regeneration

Four dpr (P12) Hippo-deficient hearts were evaluated in more depth. Fourhours prior to harvest, hearts were pulsed with EdU to visualize cellsthat had entered the cell cycle. In control hearts, EdU-positive cellswere primarily found in the GFP negative, non-cardiomyocyte lineage nearthe resected zone and most likely are infiltrating inflammatory cellsand proliferating cardiac fibroblasts. Similar proliferatingGFP-negative cells were also observed in Salv CKO hearts. In contrast tocontrols, Salv CKO resected hearts had EdU/GFP double positivecardiomyocytes within both the border zone and distal heart regions(Heallen et al., 2013; Morikawa et al., 2014).

Hippo-deficiency enhances the ability of cardiomyocytes to re-enter thecell cycle throughout the whole heart. In Salv CKO mutant hearts, cellsderived from the cardiomyocyte lineage detached from surrounding borderzone cardiomyocytes and entered the resected zone that contained a largenumber of non-cardiomyocyte cells. Moreover, Hippo-deficientGFP-positive myocardial derived cells extended lamellipodia-likeprotrusions. In control hearts, GFP positive cells grouped togetherwithin the border zone and did not infiltrate the resected region of theheart (Morikawa et al., 2014).

Example 4 Yap Directly Regulates Genes that Promote Cell CycleProgression and Cytoskeletal Remodeling

To gain further insight into the direct targets of Hippo-signaling incardiac regeneration, chromatin immunoprecipitation sequencing(ChIP-seq) experiments were performed using an antibody against theHippo effector Yap. We used mRNA expression data from microarrayexperiments to overlay the ChIP-seq dataset with upregulated genes inNkx2.5^(cre) Salv mutant hearts (FIGS. 2A-2B). Gene expression changeswere validated by qRT-PCR experiments (FIG. 2B). Gene Ontology analysisindicated that Yap directly regulates genes involved in cell cycleprogression and cytoskeletal dynamics (FIG. 2A and (Morikawa et al.,2014)). Also included among Yap regulated cell cycle genes are cyclindependent kinases, such as Cdk6, and the previously validated Yaptarget, CyclinE2. Another Yap target, the cell cycle gene Lin9, is amember of the MuvB complex, which enhances the G2/M transition(Kleinschmidt et al., 2009; Sadasivam et al., 2012) (FIGS. 2A-2B).

Yap target genes regulating both cytoskeletal and cell cycle progressioninclude Aurkb and Birc5 (survivin) (FIGS. 2A-2B). Both Aurkb and Birc5are chromosome passenger complex components and are important forchromosome condensation and segregation during mitosis, as well as forcytokinesis. Importantly, Birc5 was shown previously to be regulated byHippo-signaling in the developing heart (Heallen et al., 2011). Genesexpressed in the cytokinetic furrow and spindle midzone that regulatecytokinesis, such as Anillin, Pkp4, and Ect2 are direct Yap targetgenes, indicating that Yap promotes cytokinesis in regeneratingcardiomyocytes (Hesse et al., 2012; Matthews et al., 2012; Wolf et al.,2006).

Example 5 Yap Directly Regulates Genes that Promote CytoskeletalRemodeling and Cell Motility During Cardiac Regeneration

Consistent with the dramatic changes in cell morphology, we found thatYap also directly binds genes that regulate the actin cytoskeleton(FIGS. 2A-2B). A number of Yap regulated targets are known to localizeto lamellipodia and filopodia such as Enah, an Ena/VASP actin regulatorthat causes cardiac dysfunction when disrupted in mice (Mejillano etal., 2004; Morikawa et al., 2014).

Yap also binds genes involved in force transmission betweencardiomyocytes and the extracellular matrix (ECM). These include genesthat are implicated in connecting actin cytoskeleton to the cytoplasmicmembrane. Sarcoglycan delta (Sgcd) and Syntrophin B1 (Sntb1) are bothcomponents of the dystrophin glycoprotein complex (DGC), which isimportant for connecting the actin cytoskeleton to the ECM and maytransmit force between muscle cells (Barton, 2006; Goyenvalle et al.,2011). Both genes are mutated in human patients with muscular dystrophyand stabilize the plasma membrane in response to mechanical stress. Theactin binding protein, Talin2, connects the actin cytoskeleton tointegrins and the ECM. Lastly, Ctnna3, a cadherin-associated geneexpressed in the intercalated disc (ICD), connects the actincytoskeleton to both the ICD and the ECM and likely senses tensionbetween cardiomyocytes (Li et al., 2012).

Example 6 Hippo Depletion Rescues the Cardiac Regeneration Defect in MDXMutant Hearts

To determine whether Hippo pathway loss of function could suppress themdx failed regeneration phenotype, we generated Salv; mdx double mutanthearts and performed apex resection at postnatal day 8 on double mutantsand control samples. As shown in FIG. 3, the Salv CKO and Salv; mdxdouble mutants regenerated the myocardium in contrast to the mdx mutantsthat cannot regenerate myocardium (Morikawa et al., 2014). Thisindicates that Hippo depletion, with resultant Yap upregulation and Yaptarget gene upregulation, can suppress the mdx heart phenotype. Ourfindings indicate that Hippo depletion in myocardium is a promisingapproach to treat DMD cardiomyopathy.

Example 7 Hippo Deletion Promotes Cardiomyocyte Regeneration in theContext of Established Heart Failure

It was determined whether Hippo pathway inactivation three weeks aftermyocardial infarct still promotes cardiomyocyte regeneration withimproved cardiac function. This is a clinically important question sincemany patients suffer from chronic infarcts that lead to pathologiccardiac remodeling with heart failure and death. Previous to this it wasunknown whether Hippo deletion could effectively promote heartregeneration in the context of an established mature scar. A myocardialinfarct was introduced into the hearts of control and uninjected SalvCKO hearts at time point zero (FIG. 4). The uninjected Salv CKO micestill express control levels of Salv. Sham controls were alsoestablished at time zero. Three weeks after infarction all mice werecarefully studied by echocardiography and operated mice that were inheart failure with reduced EF were included in the study and received atamoxifen injection to inactivate the Hippo pathway.

Cardiac function parameters were evaluated in all hearts at two-weekintervals after tamoxifen injection (FIG. 4). The Salv CKO mutants hadfunctional recovery starting at the two-week timepoint (5 weeks post MI)and function continued to improve until it reached the level ofun-operated controls (9 weeks post MI). Hippo pathway inactivation afterestablished heart failure results in cardiac repair with return ofcardiac function.

Example 8 Design of Small Hairpin RNAs (shRNA) to Inactivate the HippoPathway in the Adult Heart

Three examples of shRNAs against Salv in regions of the molecule thatare conserved between mouse, human, and swine so that they can beinterchangeably used between these three species using standard methods((Tafer, 2014); FIGS. 6, 7, and 8). The functional efficiency of eachshRNA was validated, using siRNA knockdown experiments, to suppressendogenous Salv expression in neonatal cardiomyocytes (FIG. 5). Allthree shRNAs effectively knocked down Salv expression approximately 70%with shRNA #3 providing the greatest knockdown efficiency.

REFERENCES

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. An isolated synthetic nucleic acid compositioncomprising: (i) a sequence comprising SEQ ID NO: 10 or a derivativenucleic acid comprising at least 80% identity to SEQ ID NO: 10, and anantisense sequence thereof; and/or (ii) a sequence comprising SEQ ID NO:11 or a derivative nucleic acid comprising at least 80% identity to SEQID NO: 11, and an antisense sequence thereof; and/or (iii) a sequencecomprising SEQ ID NO: 12 or a derivative nucleic acid comprising atleast 80% identity to SEQ ID NO: 12, and an antisense sequence thereof;wherein when the sequence and the antisense sequence are hybridizedtogether to form a duplex structure, the sequence and the antisensesequence are separated by a loop structure.
 2. The composition of claim1, wherein the derivative nucleic acid is at least 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to one of SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO:
 12. 3. The composition of claim 1, wherein said nucleic acidis at least 43 nucleotides in length.
 4. The composition of claim 1,wherein said nucleic acid is no more than 137 nucleotides in length. 5.The composition of claim 1, wherein the loop structure is between 5 and19 nucleotides in length.
 6. The composition of claim 1, wherein thederivative nucleic acid has 1, 2, 3, 4, or 5 mismatches compared to therespective SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
 12. 7. Thecomposition of claim 1, wherein the nucleic acid or derivative nucleicacid is comprised in a vector.
 8. The composition of claim 7, whereinthe vector is a viral vector.
 9. The composition of claim 8, wherein theviral vector is an adeno-associated viral (AAV) vector.
 10. Thecomposition of claim 9, wherein the viral vector is an AAV9 vector. 11.The composition of claim 7, wherein the nucleic acid is operably linkedto a tissue-specific or cell-specific promoter.
 12. The composition ofclaim 11, wherein the promoter is a cardiomyocyte-specific promoterselected from the group consisting of: a rat ventricle-specific cardiacmyosin light chain 2 (MLC-2v) promoter; a cardiac muscle-specific alphamyosin heavy chain (MHC) gene promoter; a cardiac cell-specific minimumpromoter from −137 to +85 of NCX1 promoter; a chicken cardiac troponin T(cTNT); and a combination thereof.
 13. The composition of claim 7,wherein two or more of nucleic acids comprising SEQ ID NO: 10, SEQ IDNO: 11, or SEQ ID NO: 12 are present on the same vector.
 14. Thecomposition of claim 13, wherein the two or more nucleic acids areregulated by the same regulatory sequence.
 15. The composition of claim14, wherein each of the two or more nucleic acids is regulated by adifferent regulatory sequence.
 16. A method of promoting cardiomyocyteregeneration in an individual, the method comprising administering tothe individual an effective amount of the composition of claim
 1. 17.The method of claim 16, wherein the heart of the individual hascardiomyocyte apoptosis, necrosis, and/or autophagy.
 18. The method ofclaim 17, wherein the individual has a cardiac condition selected fromthe group consisting of: cardiovascular disease, cardiomyopathy, heartfailure, myocardial infarction, ischemia, necrosis, fibrosis, diabeticcardiomyopathy, and age-related cardiomyopathy.
 19. The method of claim17, wherein the composition is administered locally to the heart of theindividual.